The overall objective of this proposal is to elucidate the biochemical mechanisms responsble for coordinating energy producing and energy utilizing processes in cardiac muscle. Studies will be conducted mainly with the working perfused rat heart and with isolated cells and isolated mitochondria. In addition, use will be made of a rapid cell disruption technique being developed in this laboratory to measure cytosolic and mitochondrial metabolites and to study Ca ion and Mg ion distributions in the cell under normal conditions and when the mitochondrial membrane potential is lowered under pathological conditions of energy deficiency. The control of respiration in mitochondria and intact heart tissue will be studied on a fundamntal level by measuring changes of the redox state of the electron transport components in relation to the phosphorylation potential of the adenine nucleotides and the proton electrochemical gradient across the mitochondrial membrane. Regulation of the transport and metabolism of leucine, valine and isoleucine and their alpha-ketoacids will be studied in perfused hearts and isolated mitochondria. Uptake of 14C-labeled branched chain amino and alpha-ketoacids will be compared quantitatively with production of 14CO2 and intermediary metabolites. The location of regulatory sites will be investigated by assay of the individual CoA and carnitine esters in tissue samples. Effects of pyruvate and fatty acid availability, and of dietary status of the rats will be explored. Effects of branched chain amino and alpha-ketoacids on pyruvate dehydrogenase flux and activity, and on the enzymes of the citric acid cycle will also be studied. The cause of heterogeneous tissue oxygenation in the perfused rat heart in the hypoxic state will be investigated from direct observations of the microcirculatory ststem. The objective is to define which vessels are responsible for the uneven pattern of coronary perfusion during severe hypoxic conditions and to determine the factors which regulate these vessels. Pharmacological agents will be used to evaluate regulatory mechanisms.